No / Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in ¿-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12¿15-fold increase in ¿7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the ¿4 nAChR subunit was not altered. These data point to the ¿7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating ¿7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).

The experiment tested the hypothesis that the acetylcholine (ACh) release in the rat auditory cortex is greater in rats undergoing auditory classical conditioning compared to rats in a truly random control paradigm where no associative learning takes place and that this is mediated by prefrontal afferent projections influencing the nucleus basalis magnocellularis (NBM), which in turn modulates ACh release in neocortex. Rats with bilateral ibotenic acid lesions of medial prefrontal and agranular insular cortices were tested in an auditory classical conditioning task while ACh was collected from the primary auditory cortex. It was hypothesized that lesions of these prefrontal areas would prevent learning-related increases of ACh release in the primary auditory cortex. The hypothesized results were supported. Results from this experiment provide unique evidence that medial prefrontal cortex projections to the NBM are important for mediating cortical ACh release during associative learning.

In situ hybridization was used to examine effects of monocular enucleation on nicotinic acetylcholine receptor subunit cc3 mRNA in the rat dLGNand visual cortex. After 28 days postoperative, there were no significant differences in α3 mRNA density between the contralateral (deprived) and ipsilateral (non-deprived) sides. The lack of obvious effects of visual deprivation on α3 mRNA density suggests that other factors, possibly intrinsic to dLGNand visual cortex, govern the postnatal expression of α3 mRNA.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative dont les premiers symptômes apparaissent généralement vers 60 ans. Elle affecte sélectivement le système moteur et provoque une paralysie progressive amenant au décès du patient par défaillance respiratoire en quelques années. À ce jour, il n’existe aucun traitement curatif, d’où la nécessité de comprendre la physiopathologie de la SLA. Bien que de nombreuses altérations dans le muscle aient été mises en évidence, sa contribution dans la SLA reste à définir. Nous avons montré que FUS est enrichi dans les noyaux sous-synaptiques de façon dépendante de l’innervation. De plus, FUS se lie au promoteur des récepteurs de l’acétylcholine et induit leur transcription de façon dépendante d’ERM. Le mutant FUS, quant à lui, est enrichi dans les noyaux extra-synaptiques et entraîne une toxicité musculaire responsable de l’altération de la jonction neuromusculaire (JNM). Au-delà de la JNM, FUS active MEF2A, de façon dépendante de PRMT1 afin de réguler les fonctions mitochondriales et la différenciation musculaire. La toxicité musculaire de FUS joue donc un rôle clé dans la physiopathologie de la SLA. / Amyotrophic lateral sclerosis is a neurodegenerative whose first symptoms generally appear around age 60. It is characterized by progressive motor neuron degeneration, paralysis and leading to death due to respiratory failure in a few years. Currently, there is no cure so the understanding of ALS physiopathology is necessary. Although many alterations in the muscle have been highlighted, its contribution in ALS remains to be defined. We showed that FUS is enriched in subsynaptic nuclei and this enrichment depended on innervation. Besides, FUS binds directly acetylcholine receptors (AchR) promoter and is required for Ermdependent induction of AChR expression. Conversely, mutant FUS is enriched on extra-synaptic nuclei and induce muscle intrinsic toxicity responsible for neuromuscular junction (NMJ) alteration. Beyond NMJ, FUS is required for muscle mitochondrial function and muscle differentiation through PRMT1-dependent MEF2A activation. Thus, FUS muscular toxicity plays a key role in the ALS physiopathology.

Nicotine has multiple behavioral effects as a result of its action in the central nervous system. Nicotine strengthens the behaviors that lead to nicotine administration (primary reinforcement), and this effect of nicotine depends on mesotelencephalic systems of the brain that are critical to goal directed behavior, reward, and reinforcement. Nicotine also serves as a ‘reinforcement enhancer’ – drug administration enhances behaviors that lead to other drug and nondrug reinforcers. Although the reinforcement enhancing effects of nicotine may promote tobacco use in the face of associated negative health outcomes, the neuroanatomical systems that mediate this effect of nicotine have never been described. The ventral tegmental area (VTA) is a nucleus that serves as a convergence point in the mesotelencephalic system, plays a substantial role in reinforcement by both drug and nondrug rewards and is rich in both presynaptic and postsynaptic nicotinic acetylcholine receptors (nAChRs). Therefore, these experiments were designed to determine the role of the VTA and nAChR subtypes in the reinforcement enhancing effect of nicotine. Transiently inhibiting the VTA with a gamma amino butyric acid (GABA) agonist cocktail (baclofen and muscimol) reduced both primary reinforcement by a visual stimulus and the reinforcement enhancing effect of nicotine, without producing nonspecific suppression of activity. Intra-VTA infusions of a high concentration of mecamylamine a nonselective nAChR antagonist, or methylycaconitine, an α7 nAChR antagonist, did not reduce the reinforcement enhancing effect of nicotine. Intra-VTA infusions of a low concentration of mecamylamine and dihydro-beta-erythroidine (DHβE), a selective antagonist of nAChRs containing the *β2 subunit, attenuated, but did not abolish, the reinforcement enhancing effect of nicotine. In follow-up tests replacing systemic nicotine injections with intra-VTA infusions (70mM, 105mM) resulted in complete substitution of the reinforcement enhancing effects – increases in operant responding were comparable to giving injections of systemic nicotine. These results suggest that *β2-subunit containing nAChRs in the VTA play a role in the reinforcement enhancing effect of nicotine. However, when nicotine is administered systemically these reinforcement enhancing effects may depend on the action of nicotine at nAChRs in multiple brain nuclei.

Alzheimer’s disease is a progressive neurodegenerative disorder generally affecting people above the age of 65 years. Even though the pathophysiological hallmarks of AD were established more than a hundred years ago, there is yet to be a drug that can stop its characteristic neuronal damage. Of the five currently FDA-approved drugs, galantamine has a unique mechanism of action. Apart from being an AChE inhibitor, galantamine can effectively potentiate (positive allosteric modulator) the effect of agonists at nAChRs at concentrations lower than those required for its action as an AChE inhibitor. Perhaps the clinical benefits observed with galantamine are associated mainly with its nAChRs-PAM action and not its AChE inhibitory effect. Inhibiting AChE causes a delay in the degradation of ACh and a prolonged presence of ACh might act at either nAChRs or mAChRs. By indirectly targeting mAChRs as well, AChE inhibitors may lead to potential side effects. Hence there is a need for specific nAChR agents. The aim of this study was to identify the structural features of galantamine that contribute solely towards its a7 nAChR-PAM effect. In doing so, we wish to divorce the structural features that might be important for interacting with AChE. Using the deconstruction approach, we have synthesized structurally abbreviated analogs of galantamine. To study the probable interactions, we docked these molecules in human a7 nAChR homology models. Ultimately, it is of interest to determine which analogs retain the PAM activity of galantamine and to address that, a preliminary screening was performed with a select few analogs using the two-electrode voltage clamp technique

Spinal cord injuries (SCI) sever communication between supraspinal centers and the central pattern generator (CPG) responsible for locomotion. Because the CPG is intact and retains the ability to initiate locomotor activity, it can be accessed electrically and pharmacologically. The goal of this thesis was to identify and characterize a novel spinal cord surface site along the sacral dorsal column (sDC) for electrically evoking locomotor-like activity in the neonatal rat spinal cord. Stimulation of the sDC robustly activated rhythmic left-right alternation in flexor-related ventral roots that was dependent on the activation of high-threshold C fiber afferents. The C fibers synapsed onto spinal neurons, which project to the lumbar segments as part of a pathway dependent on purinergic, adrenergic, and cholinergic receptor activation. In ventral roots containing only somatic efferents, rhythmic activity was rarely recruited. However, in ventral roots containing both autonomic and somatic efferents, sacral dorsal column stimulation recruited autonomic efferent rhythms, which subsequently recruited somatic efferent motor rhythms. The efferent rhythms revealed a half-center organization with very low stimulation frequencies, and the evoked alternating bursts entrained to the stimuli. Similar entrainment was seen when sDC stimuli were applied during ongoing neurochemically-induced locomotor rhythms. The rhythmic patterns evoked by sDC stimulation operated over a limited frequency range, with a discrete burst structure of fast-onset, frequency-independent peaks. In comparison, neurochemically-induced locomotor bursts operated over a wide frequency range and had slower time to peaks that varied with burst frequency. The overall findings support the discovery of an autonomic efferent pattern generator that is recruited by sacral visceral C fiber afferents. It is hoped that this research will advance the understanding of afferent activation of the lumbar central pattern generator and potentially provide insight useful for future development and design of neuroprosthetic devices.

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Mirtazapine is an atypical antidepressant employed clinically for the treatment of major depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs). serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions at the α2a-AR have been proposed to play a role in its putative earlier onset of action. However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a- ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine, as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR functions. Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H), a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo) were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine, yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a- AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation respectively. The results from the current study show that mirtazapine binds to the α2a-AR with an affinity value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells. Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y cells does not alter muscarinic receptor function (different from fluoxetine and imipramine), but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells (typically associated with chronic antidepressant activity). Although inconclusive, the data also suggests that mirtazapine may reduce α2a-AR function. / Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.

Orientador : Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-03T23:03:55Z (GMT). No. of bitstreams: 1 Mendes_ZarifTorresRehder_M.pdf: 784934 bytes, checksum: dac9ec18f5f84c47d0194a64020fd1d8 (MD5) Previous issue date: 2004 / Resumo: No presente trabalho estudamos o padrão de distribuição dos receptores de acetilcolina (AChRs) e terminais nervosos em animais C57BL/10, após longo período de regeneração muscular. A degeneração-regeneração muscular foi induzida pelo anestésico local cloridrato de lidocaína no músculo esternomastóideo. Após 90 e 150 dias, os músculos foram retirados e os AChRs e terminais nervosos marcados com rodamina-a-bungarotoxina e anti-neurofilamento, respectivamente e observados através de microscopia de fluorescência confocal. Em todas as junções superficiais da fibra regenerada observada (n=416 junções) os receptores estavam distribuídos em ilhas e a fibra muscular apresentava núcleo central, caracterizando sua regeneração. Os terminais possuíam finas arborizações com dilatações em suas extremidades, situadas no centro das ilhas dos receptores. O padrão de distribuição da acetilcolinesterase foi semelhante ao observado para os AChRs. Nas fibras musculares regeneradas e desnervadas (n=900 junções) os AChRs não estavam distribuídos em ilhas. Esses resultados mostram que os receptores colinérgicos, após longo tempo de regeneração muscular, mantêm o padrão de distribuição em ilhas e o terminal nervoso parece determinar este padrão, provavelmente em consequência de brotamentos intraterminais que ocorrem em presença de regeneração muscular / Abstract: Mdx mice have deficiency of dystrophin and cycles of muscle fiber regeneration. Changes in the distribution of acetylcholine receptors have been reported at the neuromuscular junction of mdx and may be a consequence of muscle fiber regeneration. At the present, we verified whether the distribution of receptors is still altered in long-term regenerated muscle fibers from C57Bl/10 mice. The left sternomastoid muscle of adult mice was injected with 60 µl of lidocaine hydrochloride to induce degeneration-regeneration. Some animals had the STN denervated at the time of lidocaine injection. After 90 and 150 days, receptors were labeled with rhodamine-a-bungarotoxin for confocal observation. For both periods studied, receptors were distributed into clusters. In denervated-regenerated fibers, receptors were distributed as branches, similar to normal denervated muscles. This suggests that nerve-dependent mechanisms are involved in the changes in receptor distribution seen in regenerated muscle fibers, after lidocaine treatment and a similar phenomenon might explain the changes in receptor distribution seen in dystrophic muscle fibers / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural